Biotechnology Review (Bio Final Review)

Biotechnology

-Biotechnology is the manipulation of organisms or their components to make useful products

-For thousands of years, humans have used microbes to make wine and cheese and selectively bred stock, dogs, and other animals.

-DNA technology is the set of modern techniques used to study and manipulate genetic material

-Genetic engineering involves manipulating genes for practical purposes

-Gene cloning leads to the production of multiple, identical copies of a gene-carrying piece of DNA

-Recombinant DNA is formed by joining nucleotide sequences from two different sources

Steps in cloning a gene

1. Plasmid DNA is isolated2. DNA containing the gene of interest is isolated3. Plasmid DNA is treated with a restriction enzyme that cuts in one place, opening the circle4. DNA with the target gene is treated with the same enzyme and many fragments are produced5. Plasmid and target DNA are mixed and associate with each other6. Recombinant DNA molecules are produced when DNA ligase joins plasmid and targets segments

together7. The recombinant plasmid containing the target gene is taken up by a bacterial cell8. The bacterial cell reproduces to form a clone, a group of genetically identical cells descended

from a single ancestral cell.

-Restriction enzymes cut DNA at specific sequences.

-Each enzyme binds to DNA at a different restriction site

-many restriction enzymes make staggered cuts that produce restriction fragments with single- stranded ends called “sticky ends.”

-fragments with complementary sticky ends can associate with each other, forming recombinant DNA.

-DNA ligase joins DNA fragments together

Eukaryote mRNA processing

-Before leaving the nucleus, mRNA is modified

-Extra nucleotides added to the ends, forming a cap (G) at one end and a tail (50 to 250 A’s) at the other

-RNA splicing: noncoding stretches removed and coding portion spliced back together

-Introns: intervening sequences-removed

-Exons: coding regions-spliced back together

Reverse transcriptase

-Complementary DNA (cDNA) can be used to clone eukaryotic genes

-Reverse transcriptase produces a DNA strand from mRNA.

-Nucleic acid probes bind very selectively to cloned DNA

DNA Profiling and Genomics

DNA Profiling

-DNA profiling is the analysis of DNA fragments to determine whether they come from the same individual

-It compares genetic markers from noncoding regions that show variation b/t individuals and involves amplifying (copying) of markers for analysis

-The Process

-DNA is first isolated

-The DNA of selected markers is amplified

-The amplified DNA is compared

PCR

-PCR: Polymerase chain reaction, is a method of amplifying a specific segment of a DNA molecule

-PCR relies upon a pair of primers that are

-short,

-chemically synthesized, single stranded DNA molecules, and

-complementary to sequences at each end of the target sequence

-PCR

-is a three-step cycle that

-doubles the amount of DNA in each turn of the cycle

-The advantages of PCR include

-the ability to amplify DNA from a small sample,

-obtaining results rapidly, and

-a reaction that is highly sensitive, copying only the target

Gel electrophoresis

-Gel electrophoresis can be used to separate DNA molecules based on size as follows:

1. A DNA sample is placed at one end of a porous gel

2. Current is applied and DNA molecules move from the negative electrode toward the positive electrode

3. Shorter DNA fragments move through the gel matrix more quickly and travel farther through the gel

4. DNA fragments appear as bands, visualized through staining or detecting radioactivity or fluorescence

5. Each band is a collection of DNA molecules of the same length

-DNA profiling is used to

-determine guilt or innocence in a crime,

-settle questions of paternity

-identify victims of accidents, and

-probe the origin of nonhuman materials

-A single nucleotide polymorphism (SNP) is a variation at a single base pair within a genome

-Restriction fragment length polymorphism (RFLP) is a change in the length of restriction fragments due to a SNP that alters a restriction site

-RFLP analysis involves

-producing DNA fragments by restriction enzymes and

-sorting these fragments by gel electrophoresis

Genomics

-Genomics is the study of an organism’s complete set of genes and their interactions

-initial studies focused on prokaryotic genomes

-Many eukaryotic genomes have since been investigated

-Genomics allows another way to examine evolutionary relationships

-Genomic studies showed a 96% similarity in DNA sequences b/t chimpanzees and humans

-Functions of human disease-causing genes have been determined by comparing human genes to similar genes in yeast.

GMO’s and Gene Therapy

-Recombinant cells and organisms constructed by DNA technologies are used to manufacture many useful products, chiefly proteins.

-Bacteria are often the best organisms for manufacturing a protein product because bacteria

-have plasmids and phages available for use as gene-cloning vectors,

-can be grown rapidly and cheaply,

-can be engineered to produce large amounts of a particular protein, and

-often secrete the proteins directly into their growth medium

-Yeast cells

-are eukaryotes,

-have long been used to make bread and beer,

-can take up foreign DNA and integrate it into their genomes

-have plasmids that can be used as gene vectors, and

-are often better than bacteria at synthesizing and secreting eukaryotic proteins

-Mammalian cells must be used to produce proteins with chains of sugars (glycoproteins). Examples include

-human erythropoietin (EPO), which stimulates the production of red blood cells,

-factorVIII to treat hemophilia, and

-tissue plasminogen activator (TPA), used to treat heart attacks and strokes

-products of DNA technology are already in use.

-therapeutic hormones produced by DNA technology (insulin, human growth hormone)

-DNA technology is used to test for inherited diseases, detect infectious agents such as HIV, and produce vaccines, harmless variants or derivatives of a pathogen that stimulate the immune system.

-Genetically modified organisms (GMO) contain one or more genes introduced by artificial means

-Transgenic organisms contain at least one gene from another species.

-GM plants are being produced that are more resistant to herbicides and pests and provide nutrients that help address malnutrition

-GM animals are being produced with improved nutritional or other qualities

GMO’s raise concerns about health and safety

-Scientists use safety measures to guard against production and release of new pathogens

-Concerns related to GM organisms include the potential introduction of allergens into the food supply and spread of genes to closely related organisms

-Regulatory agencies are trying to address the safety of GM produced foods, and safe use of biotechnology

Gene therapy

-Gene therapy aims to treat a disease by supplying a functional allele

-Gene therapy is an alteration of an afflicted individual’s genes and attempt to treat disease

-Gene therapy may be best used to treat disorders traceable to a single defective gene.

-The first successful human gene therapy trial in 2000

-tried to treat 10 children with SCID (severe combined immune deficiency)

-helped nine of these patients, but caused leukemia in three of the patients and resulted in one death

-The use of gene therapy raises many questions

Organismal Cloning

-Developing and refining methods to clone whole multicellular organisms

-production of stem cells

-Under correct circumstances, stem cells can become any kind of cell

-Great potential for growing new organs for transplantation, etc.

Cloning Plants

-Plants grown from cuttings are clones of the original

-First single cell cultures of plants in 1950s at Cornell U

-Now used extensively in agriculture

Cloning animals

-Differentiated animal cells do not divide in culture nor do they produce multiple cell types

-Nuclear transfer is the answer

-Transfer differentiated nucleus into enucleated egg cell

-Discovery: something in nucleus does change as animal cells differentiate

Cloning Mammals

-Other experiments showed some success with embryonic nuclei but not with differentiated ones

-First mammal cloned from differentiated nucleus-1997 in Scotland

-Fused nucleus with enucleated eggtransplanted embryo to surrogateDolly

-Process involved 3 different animals (cell donor, egg donor, surrogate mother)

Problems with cloned animals

-Most trials unsuccessful

-Many cloned animals exhibit defects

-Metabolic disorders

-early death

-Differences probably due to epigenetic changes—misplaced methyl groups on DNA may affect pattern of gene expression in differentiated nuclei

Stem Cells

-Most cloning of human cells directed toward production of stem cells--*undifferentiated cells that can grow into many different types of cell

-embryonic stem cells are pluripotent

-Adult stem cells less so

-With the right culture conditions, adult stem cells have been made to differentiate into different types of cells

-Cloning of human cells—especially embryonic cells—has been highly controversial

-Embryonic

-When grown under different culture conditions, may produce different types of differentiated cells

-Adult

-When grown under certain culture conditions, may produce fewer different types